Generation of 5 and 17 kDa human growth hormone fragments through limited proteolysis

Abstract

Introduction. The reported presence of two fragments of 5 and 17 kDa originating from the 22 kDa human growth hormone (hGH) in blood and tissues, postulated as the sequences AA1–43 and AA44–191, has led to the hypothesis of a post-translational proteolytic origin with respect to the abundant 22 kDa variant (AA1–191). To evaluate this hypothesis, the activity of several endo-proteases on the 22 kDa hGH protein has been evaluated. Methods. Proteolysis using pepsin, trypsin, V8-protease, proteinase K and thermolysin were explored under several conditions, including incubation time and pH. Results were monitored by MALDI-TOF and HPLC-ESI mass spectrometry. Proteolytic 5 and 17 kDa fragments were purified through reversed phase HPLC-UV, and their immuno-affinity properties evaluated by surface plasmon resonance. Results. Thermolysin was shown to target mainly the AA43–44 bond of the 22 kDa sequence at physiological pH. Interaction studies of the purified fragments with anti-GH antibodies showed some reactivity for the 17 kDa fragment. Conclusions. Thermolysin processes hGH generating 5 and 17 kDa fragments, demonstrating the feasibility of this reaction, although the enzyme responsible for this process in humans is still unknown. Specific antibodies should be used to detect these fragments in human specimens, and, at the same time, the 17 kDa fragment could constitute an interference in some hGH immunoassays.